Nucleic acid processing methods, kits and devices

ABSTRACT

Biological samples containing nucleic acids, RNA and DNA, are freed from bound proteins by incubation with a chaotropic agent such as a guanidium salt, and the mixture is readied for further processing by dilution of such agent to a concentration below 0.05 M without physical isolation of RNA and DNA from one another or from other components of the reaction mixture. Methods include such preparation and further processing, such as amplification and detection, which may be performed in a single container. Chaotropic agent may be supplied as a dried reagent adhered to a container. Kits may include such reagents, alone or with amplification reagents.

TECHNICAL FIELD

This invention relates to preparation of one or more nucleic acidmolecules, either DNA or RNA, as templates freed of bound proteins andavailable for subsequent uses including detection, modification, and/ormanipulation with our without intervening amplification of sequenceswithin said nucleic acid molecules.

BACKGROUND

Nucleic acid molecules, RNA and DNA, present in both cellular andnon-cellular samples are often associated with proteins or othermacromolecules whose binding interferes with either detection orenzymatic manipulation of the nucleic acids. For this reason mostprotocols designed to detect or utilize nucleic acids begin with one ormore purification and/or isolation steps that are carried out prior toany subsequent manipulation, such as amplification of particular targetsequences or replication of reporter sequences.

Methods used to prepare the nucleic acid must be compatible with thesubsequent biochemical steps. In addition, it is preferable to performpurification in the fewest reliable steps and the smallest possiblenumber of containers in order to reduce losses. Quantitative accuracyand convenience of use are important features of assays, which may becarried out on large numbers of small samples, for instance samplescomposed of only one cell, or a relatively small number of cells, orsamples comprised of a small piece of tissue, a fraction of a cell, or asmall volume of a cellular extract or homogenate, or non-cellularsamples of nucleic acids. Handling and processing, particularly of smallsamples, should not result in the loss, degradation, or contamination ofsaid samples or of the nucleic acids within such samples.

Traditional methods for the purification or isolation of DNA and RNAfrom cells and tissues and the separation of DNA molecules from RNAmolecules, typically include, but are not limited to: a)disruption/denaturation of the sample in the presence of strongdenaturant agents such as guanidine salts, urea, detergents, strongalkali, or a combination of the above; b) separation of nucleic acids ofinterest from denatured proteins and/or other nucleic acids byextraction with a non-aqueous liquid, such as phenol:chloroform (usedfor total RNA and DNA separation), or by absorption to a matrix, resin,beads or fibers (used for selective extraction of mRNA and for otherapplications), or by neutralization with alkaline buffer andcentrifugation (used for plasmid DNA isolation); c) recovery of nucleicacids by precipitation with an alcohol or a monovalent cation such assodium or ammonium, or lithium chloride, and re-suspension to anappropriately small volume or, alternatively, elution of nucleic acidsin an appropriately small volume. The volume in which the purifiednucleic acids are contained determines the fraction of nucleic acidsthat can be analyzed in one assay. Volume is of particular importancewhen analysis is carried out on very small samples such as single cellsor very few cells. It is known in the art that certain starting samplesrequire the use of harsher conditions to disrupt cells. Conditions canbe chosen to selectively degrade or digest DNA or RNA for recovery ofthe other. RNA molecules are much more sensitive to degradation than DNAmolecules, due to their sensitivity to alkaline conditions, to hightemperature, and to the ubiquitous presence of RNases. Thus, manyprotocols for isolation of RNA require milder conditions and include thepresence of agents designed to inhibit RNases.

Examples of known methods for genomic DNA or RNA purification,isolation, or separation include use of: cetyltrimethylammonium bromide(CTAB) and high salt concentration, (Jones, A. S. (1963), Use ofAlkyltrimethylammonium Bromides for the Isolation of Ribo- andDeoxyribo-Nucleic Acids, Nature 199:280-282); low salt concentrationunder hyperbaric, hydrostatic pressure (U.S. Pat. No. 6,111,096); gentlesalt precipitation (and optional protease digestion); irreversiblebinding to aluminum oxide-covered matrixes (U.S. Pat. No. 6,291,166);preparation of RNA by guanidinium thiocyanate lysis followed bycentrifugation through a CsCl cushion (Chirgwin, J. M. et al. (1979),Isolation of Biologically Active Ribonucleic Acid from Sources Enrichedin Ribonuclease, Biochemistry 18:5294-5299); various modifications andimprovements of the acid guanidinium thiocyanate-phenol-chloroformmethod (Chomczynski, P. and Sacchi, N. (1987), Single-Step Method of RNAIsolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction,Anal. Biochem. 162:156-159), later expanded to include DNA isolation(Chomczynski, P. (1993), A Reagent for the Single-Step SimultaneousIsolation of RNA, DNA and Proteins from Cell and Tissue Samples,Biotechniques 15:532-537); binding of total RNA or DNA to matrixes suchas glass fiber filters, silica-gel membranes, magnetic beads orcellulose-based matrixes.

Specific extraction of mRNA molecules only is performed by interactionof their poly(A) tails with oligo (dT) attached to cellulose or resinscontaining a streptavidin complex; and selective precipitation of mRNAmolecules with Poly T PNA probes and streptavidin. An intact poly(A)tail is probably not always present, particularly for very long RNAmolecules such as Xist mRNA (Hong, Y. K. et al. (1999), A New Structurefor the Murine Xist Gene and its Relationship to ChromosomeChoice/Counting During X-Chromosome Inactivation, Proc. Natl. Acad. Sci.USA 96:6829-6834).

Fluorescence in situ hybridization (FISH) is an example of isolation andpurification in situ. Procedures typically involve fixation of thesample, during which bound proteins are denatured, followed by washingsteps in the presence of a detergent that removes at least someproteins. All these manipulations necessitate a considerabletime-and-labor investment. Moreover, they all can lead to loss ofnucleic acid molecules during the purification/extraction procedure orto poor detection of the target sequences. The latter can be a problem,since FISH probes need to recognize and visualize above background avery low number of target molecules.

Recently real-time target amplification methods, for example real-timePCR techniques, have provided a powerful tool for accuratequantification of RNA copy numbers, making possible the study of finemodulations of gene expression levels. As previously mentioned, however,RNA isolation is highly challenging, because of both thephysical/chemical characteristic of this type of molecule and itssensitivity to the action of multiple RNases, present intracellularly oreasily introduced by environmental contamination. Typically DNA isremoved from RNA preparations by chemical, physical, or enzymaticmethods. All of the above manipulations have contributed to sheddingdoubt on the reliability of RNA copy estimates obtained with theavailable protocols, particularly when analyzing very small samples.(See Klein, C. A. et al. (2002), Combined Transcriptome and GenomeAnalysis of Single Micrometastatic Cells, Nat. Biotechnol. 20:387-392).

Strategies have been devised to achieve mRNA capture, reversetranscription and PCR amplification in the same vessel, thereby limitingloss or damage of nucleic acids molecules during purification. Oligo(dT)-coated multi-well plates or streptavidin-coated PCR tubes to beused in conjunction with biotin-labeled oligo (dT)₂₀, for example, areused for capture. Nucleic acids shearing may be lower with such methods,and loss of material due to transfer to a new vessel is avoided, but theaccuracy of RNA quantification still depends on the efficiency ofmRNA-binding to the capturing molecules.

Preparation of total RNA, rather than mRNA, is an alternative for thesingle-tube (or single-vessel, if microchips are used) approach,provided that removal of the proteins bound to nucleic acids is achievedin a way that doesn't interfere with subsequent steps of reversetranscription (RT) and PCR and does not affect RNA integrity.

Cell lysis by a simple freeze-thaw cycle neither separates proteins fromnucleic acids, nor inactivates cellular RNases. Lysis by boilingbacterial cells surely leads to RNA hydrolysis.

A Cells-to-cDNA II Kit from Ambion, Inc. (Austin, Tex. U.S.A.) employs aCell Lysis II Buffer compatible with RT and PCR. RNA copies can be thusamplified in one tube while DNA is degraded via DNase digestion, assuggested by the manufacturer. RT and PCR are then carried outsequentially by addition of the appropriate buffers and reagents to thelysed sample. The Cell Lysis II Buffer/RT PCR Buffer ratio tolerated bythe assay, however, is low so that only a fraction of the lysed samplecan be used for each RT-PCR assay. This technique, therefore, is notsuitable for the analysis of very small samples, comprised of few orsingle cells. Non-ionic detergents, such as Triton® X-100 or NP40, areused in a number of protocols to lyse the cells plasma membrane andrelease cytoplasmic RNA pools. These detergents, at appropriateconcentrations, are compatible with enzymatic reactions and cytoplasmicsamples prepared with this method can be directly used in RT-PCR assaysor other manipulation of RNA molecules aimed at their detection and/orquantification (Brady, G. and Iscove, N. N. (1993), Construction of cDNALibraries from Single Cells, Methods Enzymol. 225:611-623; Hansis, C. etal. (2001), Analysis of October-4 Expression and Ploidy in IndividualHuman Blastomeres, Mol. Hum. Reprod. 7:155-161). Genomic DNA and nuclearRNA, such as Xist RNA, cannot, however, be prepared with theseprocedures.

Our laboratory developed an assay capable of measuring both genomic DNAand mRNA copies of genes of interest in single mouse embryos orblastomeres (Hartshorn, C., Rice J. E., Wangh, L. J. (2002),Developmentally-Regulated Changes of Xist RNA Levels in SinglePreimplantation Mouse Embryos, as Revealed by Quantitative Real-TimePCR, Mol. Reprod. Dev. 61:425-436; Hartshorn, C., Rice, J. E., Wangh, L.J. (2003), Differential Pattern of Xist RNA Accumulation in SingleBlastomeres Isolated from 8-cell Stage Mouse Embryos Following LaserZona Drilling, Mol. Reprod. Dev. 64:41-51; Hartshorn, C., Rice, J. E.,Wangh, L. J., Optimized Real-Time RT-PCR for Quantitative Measurementsof DNA and RNA in Single Embryos and Blastomeres, In: Bustin S. A., ed.A-Z of Quantitative PCR, pages 675-702, International University Line,In press.). Counting genomic DNA copies in very small samples providesan ideal internal standard for nucleic acid recovery and for PCRefficiency. Moreover, there is no need to use DNase and the accompanyingheat-inactivation of the enzyme, during which some RNA can be hydrolyzed(RNA degradation is enhanced by the presence of magnesium, required forDNase activity). Our attempts to adapt the Ambion Cells-to-cDNA II Kitto RT-PCR of RNA and DNA from single embryos/cells failed, probably dueto the higher-than-recommended amount of Cell Lysis Buffer that had tobe used in order to assay whole specimens rather than an aliquot of thespecimen. While RNA was measured at the expected levels inhigh-expressing samples, genomic DNA, present in low copy numbers, wasvery often undetected.

Examples of lysis buffers aimed at the preparation of DNA templates onlyand compatible with PCR analysis in the same reaction vessel are knownand commercially available. Generally these methods do not allowsimultaneous/parallel analysis of DNA and RNA molecules from the samesamples, because RNA is degraded during the extraction procedure. TheRelease-IT™ (CPG, Inc., Lincoln Park, N.J., U.S.A.) is a proprietaryreagent that releases DNA from whole blood, cell cultures, bacterialcolonies and other biological samples. Lysis is accomplished directly inthe amplification tube on a thermal cycler, and PCR reagents aresubsequently added to the lysate initiating amplification. Release-IT™sequesters cell lysis products that might inhibit PCR. Unlike othermethods described above, this allows RNA recovery and reversetranscription of small aliquots of the sample for RT-PCR (the wholesample can be used for PCR of genomic DNA). However, the initial heatingcycle required for Release-IT™ action is believed to be detrimental toRNA, because it includes a total of 4 minutes at 97° C. and a holdingstep at 80° C. Moreover, the Release-IT™ reagent is not recommended foramplification of low copy number DNA without cellular enrichment.

Nucleic acids analysis in very small samples, including single cells ora portion of a single cell, presents a number of challenges. Whileseveral commercial kits offer RT-PCR sensitivity down to the single-celllevel, this claim often implies harvesting a larger sample of which onlya portion is used for each assay. A few kits promise efficient nucleicacids extraction from actual single cells, but collection of theindividual samples themselves is frequently difficult and should be donecarefully to preserve RNA content (Hartshorn, C., Rice, J. E., Wangh, L.J. (2003)). Recently, laser capture microdissection (LCM) and laserpressure catapulting (LPC) have made precise excision of single cells,or compartments of single cells, possible. Further, two techniques havealready been developed for single-cell gene expression profiling thatrely on polyadenylation of mRNA molecules for their direct detection ina cell lysate, without need for RNA purification (3 prime endamplification, or TPEA, and global amplification of cDNA copies of allpolyadenylated mRNAs, or PolyAPCR) (reviewed by Brady, G. (2000),Expression Profiling of Single Mammalian Cells—Small is Beautiful, Yeast17:211-217). Both techniques are limited to cytoplasmic RNA measurementsand do not extend to DNA or nuclear RNA.

An aspect of this invention is a method for preparing DNA or RNAmolecules, or both, for amplification and detection or for otherenzymatic processing of mixtures of DNA and RNA molecules that have beenfreed of bound proteins, such mixtures comprising freed DNA and RNAmolecules, a chaotropic agent, and degraded and denatured proteins,comprising diluting the mixture so as to reduce the concentration ofchaotropic agent to less than 0.05 M, preferably less than 0.01 M,without removing or isolating the DNA and RNA molecules from each otheror from the other components of the mixture.

Another aspect of this invention is preparing the foregoing mixture,prior to diluting it, by incubating a sample containing protein-boundDNA and RNA molecules with a concentrated disruption reagent containingat least 2 M chaotropic agent.

Another aspect of this invention is amplifying one or more DNA and RNAsequences in the diluted mixture without physically separating the DNAand RNA molecules from the diluted mixture.

A further aspect of this invention is performing the foregoing dilutionsand amplifications in a single container and, preferably, also preparingthe mixture in the container.

Yet another aspect of this invention is a device useful in freeing DNAand RNA molecules from bound proteins comprising a dry or semi-drydisruption reagent containing a chaotropic reagent that is adhered to asurface of a container or a container part, such as a cap of a tube or asurface within a multiple-chambered container.

Another aspect of this invention is kits including the foregoing deviceand additional reagents and materials for performing dilution andfurther processing of diluted mixtures, for example, amplification andsequencing reactions.

SUMMARY

This invention includes methods, devices and reagents for preparation ofdeoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules foramplification and detection or other enzymatic processing withoutphysical isolation of nucleic acids from denatured proteins or physicalisolation of one type of nucleic acids (DNA or RNA) from the other.Preparative methods according to this invention can be performed in asingle container, without any transfer of sample-containing material.Further processing, such as amplification and detection, can beperformed in the container holding the prepared nucleic acids, alsowithout any transfer of sample-containing material. Methods according tothis invention include preparation, amplification and detection ofnucleic acids in a single container, for example, a 200 μl tube suitablefor use with current thermal cycling instruments. Methods according tothis invention reduce or minimize shearing of nucleic acids by reducingor eliminating transferring, washing and resuspension of nucleic acids,thereby reducing the problem of mispriming in amplification reactions,particularly amplifications utilizing the polymerase chain reaction.

Nucleic acids sample sources to which methods of this invention can beapplied include single cells, groups of cells (embryos, tissue samples),cell lysates, or other sources of nucleic acids, for example, cytoplasmsipped from cells. If the source is one or more cells, preparationincludes cell lysis, which is not required if a lysate is the source.Samples to which methods of this invention are applied contain nucleicacids to which proteins are bound.

The first step in methods according to this invention is to free nucleicacids from bound proteins and to inactivate nucleases. If the sample iscellular, the first step also includes lysing the cell or cells. For thefirst step the sample is treated with a reagent conveniently referred toas a Disruption Reagent. The essential component of the DisruptionReagent is a chaotropic agent to denature or degrade all proteins,including nucleases. Suitable chaotropic agents include guanidium salts,such as guanidine isothiocyanate or guanidine hydrochloride, andpotassium iodide. Our preferred agent is guanidine isothiocyanate. Thefirst step is performed in a small volume, less than 10 μl of DisruptionReagent, preferably less than 1 μl and, for embodiments that includeamplification in a single tube, more preferably 20-50 nl so as to permitserial dilutions without exceeding the capacity of a standard 200 μltube. The first step is also performed at a high concentration ofchaotropic agent, sufficient to achieve at least 2 M concentration ofmonovalent cation. Working at room temperature, guanidine isothiocyanateat high concentrations tends to precipitate (and clog small-bore pipetssuitable for dispensing nanoliter quantities). To permit working with apreferred concentration of 4 M guanidine isothiocyanate, for example, weadd one percent or more by volume of dimethylsulfoxide (DMSO), awater-miscible solvent that prevents precipitation but can be evaporatedduring a subsequent drying step. If the Disruption Reagent is not driedprior to use in processing the nucleic acids, the presence ofdimethylsulfixide has the added benefit of enhancing the permeability ofcell membranes to external chemicals, such as the chaotropic agent.

As indicated earlier, lysing is needed in the first step, if the sampleis one or more cells. The Disruption Reagent may include a detergent forthis purpose. We have used sarcosyl, obtained from Stratagene, La Jolla,Calif., U.S.A., as the detergent. Additional ingredients that may beincluded in the Disruption Reagent include a reducing agent, preferablyone that evaporates during drying, such as βmercapto-ethanol, achelating agent, and a neutral buffer, such as sodium citrate.

The Disruption Reagent may be used as a liquid, or it may be dried andreconstituted during use. We have successfully dried and thenreconstituted to its original volume 20 nl of the following DisruptionReagent: 0.25% sarcosyl (detergent), 2 M guanidine isothiocyanate(chaotropic agent), 1% (vol/vol) DMSO (solvent), 100 mMβmercapto-ethanol (reducing agent) and 0.01 M sodium citrate, pH 7.0(buffer).

The first step in methods according to this invention comprisesincubating a sample with a Disruption Reagent for a time and temperaturesufficient to permanently denature proteins that are present. Inpreferred embodiments the first step includes heating to concentrate themixture by evaporation. This raises the concentration of chaotropicreagent significantly, thereby ensuring permanent protein denaturation.Although not preferred, the initial concentration of chaotropic reagentcan be reduced, because, if heating is included, evaporation willconcentrate the agent to or above the required 2 M concentration. Mostpreferably, heating is performed promptly after the sample andDisruption Reagent (reconstituted to original volume, if dried) aremixed. It is preferred that heating be sufficient to evaporate themixture to a semi-dry (moist solid) or dry product. Such heating willevaporate DMSO and βmercapto-ethanol, if present. The duration andintensity of heating is adjusted experimentally so as to prevent loss ofnucleic acid target sequence(s) of an assay or at least prevent lossfrom exceeding 25 percent. Dry and semi-dry product from the first stepcan be used immediately or stored pending further processing.

The second step in methods according to this invention is to dilute theliquid, dry or semi-dry product from the first step, either prior to oras part of further processing of nucleic acids in the mixture. Forpurposes of illustration, we will describe in detail processing of freednucleic acids beginning with reverse transcription (RT). It is importantthat prior to or at least by this first enzymatic step, dilution of themixture resulting from the first step be sufficient so that reagents,particularly including chaotropic agent, in the mixture at this pointare reduced to a concentration that is so low that they do notsignificantly adversely affect the processing. In all cases the amountof dilution for further processing must be sufficient to lower theconcentration of chaotropic agent to less than 0.05 M. For theillustrative processing of reverse transcription, we have found thatdilution should reduce the concentration of our preferred chaotropicagent, guanidine isothiocyanate to below 0.05 M, preferablysignificantly lower, more preferably below 0.01 M. As can be seen fromthe Examples, we have successfully utilized dilution to 0.004 Mguanidine isothiocyanate. The required degree of dilution for particularprocessing can readily be determined experimentally. In embodiments thatare performed by hand, we prefer to utilize dilution volumes of at least1 μl, as smaller volumes are difficult to pipet accurately. That willnot be the case with chip-type embodiments, which may include precisemachine control, and we prefer in such embodiments to minimize volumesto lower device and reagent costs to the extent practicable.

As indicated, dilution of the second step can be performed either as asingle dilution or as a series of dilutions. For illustration,proceeding to reverse transcription can be performed in two dilutions:first adding water containing primers for reverse transcription, heatingto melt double strands and then cooling to anneal RT primers; followedby a second dilution with reverse transcriptase in RT buffer. Using thisprocedure, we prefer that the first dilution itself be at least 50:1,more preferably higher, for example at least as high as 300:1; and thatthe second dilution, including revere transcriptase and RT buffer,complete the dilution. Also, as discussed in more detail below, otherappropriate enzyme treatments (for example, DNase or a glycosidase suchas cellulase, amylase, β-glucosidase or lysozyme) can be performed withdilution, either a single dilution or a series of dilutions. In theExamples we demonstrate enzyme activity with dilutions of 200:1 and500:1.

Following completion of the second step, the sample is prepared andready for further processing. Further processing may includeamplification-and-detection assays. Such assays may include exponentialamplification of target molecules or reporters, utilizing, for example,methods known in the art, such as the polymerase chain reaction (PCR)process (U.S. Pat. Nos. 4,683,202, 4,683,195, 4,965,188), nucleic acidsequence-based amplification (Heim, A. et al. (1998), Highly SensitiveDetection of Gene Expression of an Intronless Gene: Amplification ofmRNA, but not Genomic DNA by Nucleic Acid Sequence Based Amplification(NASBA), Nucleic Acids Res. 26: 2250-2251, strand-displacementamplification (SDA), transcription-mediated amplification (TMA),rolling-circle amplification (RCA) (Daubendiek, S. L. and Kool, E. T.(1997), Generation of Catalytic RNAs by Rolling Transcription ofSynthetic DNA Nanocircdes, Nat. Biotechnol. 15: 273: 273-277), andramification amplification methodology (RAM). Because PCR is the mostwidely used amplification technique, it is used herein for explanatorypurposes. Persons skilled in the art will be able to apply theexplanations to other amplifications techniques.

Selected sequences of the nucleic acids, both RNA and DNA resulting fromthe second step may be further processed by a PCR amplification assay.To amplify RNA, it is first converted to cDNA by reverse transcription.Following that, DNA—both genomic DNA and cDNA—can be amplified. Onepreferred method is to heat the mixture from the second step to meltdouble-stranded nucleic acid sequences and then to cool the mixture soas not to denature reverse transcriptase enzyme. Reverse transcriptaseenzyme and its buffer are then added to the mixture of the second step,along with primers, if primers are not already present. In carrying outreverse transcription, we prefer not to further dilute the product morethan necessary, preferably not more than 6:1 and even more preferablynot more than 2:1 for the reason stated above.

At this point one may optionally eliminate RNA from the mixture. RNAmolecules may interfere with certain processing of genomic DNA and cDNA.For this purpose we add RNase H to the mixture, so as to degrade RNAfrom RNA: cDNA hybrids. Again, we prefer to minimize the increase involume from this addition, and we prefer to limit the increase to 5%.The mixture is incubated for a time and temperature sufficient for RNasedigestion of RNA strands in the hybrids.

Next we add to the mixture a PCR amplification mixture containing allnecessary reagents to amplify one or more selected target sequences bythe polymerase chain reaction process, including all primers,precursors, polymerase enzyme, controls and PCR buffer. The polymerasechain reaction process may be a traditional symmetric PCR amplification,utilizing forward and reverse primers in equimolar amounts; anasymmetric PCR amplification, utilizing one primer in excess; or aLATE-PCR amplification, as disclosed in pending U.S. patent applicationSer. No. 10/320,893, filed Dec. 17, 2002 and in published internationalpatent application WO 03/054233. Asymmetric PCR and LATE-PCR bothinclude exponential amplification followed by linear amplification.Alternative PCR-based amplification methods may also be used, forexample, Serial Analysis of Gene Expression (SAGE). (Velculescu, V. E.et al. (1995), Serial Analysis of Gene Expression, Science 270:484-487). Persons skilled in the art will appreciate that reagents forother amplification systems are added at appropriate steps in theprocess. For rolling circle amplification, for example, one may add oneor more reporter sequences to the mixture in the second step, andperform enzymatic ligation in place of reverse transcription in thesubsequent step.

Amplification assays may be end-point assays or real-time assays whereinthe amplification mixture includes at least one reporter, for example,an intercalating dye such as SYBR green or dual-labeled fluorescenthybridization probes such as end-labeled linear probes for the 5′nuclease assay (U.S. Pat. No. 5,538,848), molecular beacon probes (U.S.Pat. No. 5,925,517), FRET probe pairs or yin-yang probes. Li, Q. et al.(2002), “A New Class of Homogeneous Nucleic Acids Probes Based onSpecific Displacement Hybridization,” Nucl. Acid. Res. 30: (2)e5).Probes may double as primers (Nazarenko, I. et al. (1997), A Closed TubeFormat for Amplification and Detection of DNA Based on Energy Transfer,Nucl. Acids Res. 15: 2516-2521).

Amplification methods may also be used to generate quantities ofdouble-stranded or single-stranded nucleic acid products for furtherprocessing, for example, sequencing, fragment analysis, or use in anassay such as the oligonucleotide ligation assay (OLA).

The Disruption Reagent may be prepared and supplied in dried form, forexample a spot (which we call a “LysoDot”) or a film (which we call a“LysoFilm”) on the surface of a container or container portion to beused for preparing nucleic acids. A LysoDot or LysoFilm of DisruptionReagent may be applied to a tube, well, or cover, such as a lid, top orcap, of the container. The container may be a PCR tube, amicrocentrifuge tube, a microtiter plate or a chip with wells, forexample. The Disruption Reagent may be supplied in liquid form,preferably including a solvent such as DMSO, in a fine-barreled pipetsuitable for dispensing small volumes, particularly nanoliter amounts.The Disruption Reagent may also be supplied as a dried pellet or as acomposition dried onto a solid support, such as a fiber or a matrix or abead that is inert and does not bind nucleic acids.

A chip or other microfluidic device may be constructed for carrying outthe first and second steps of nucleic acid preparation in differentchambers, and may include one or more additional chambers for subsequentprocessing.

Reagent kits may include dried Disruption Reagent, preferably LysoFilm-or LysoDot-containing containers or container portions, in combinationwith all or some reagents for performing at least one enzymatic process.Kits may include, for example, some or all RT reagents (primers, dNTPs,buffer, enzyme, RNase inhibitor, reducing agent). A DNase may beincluded. Similarly, some or all amplification reagents may be included(primers, dNTPs, buffer, polymerase), as may detection reagents(hybridization probes, which may be dual-labeled fluorescenthybridization probes, or intercalating fluorescent dye) or reagents forpost-amplification processing, for example sequencing (enzyme,sequencing primer). Other enzymes may be included as appropriate.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph of results of PCR assays for a first target sequenceperformed on replicate nucleic acid samples prepared according to thisinvention.

FIG. 2 is a graph of results of PCR assays for a second target sequenceperformed on replicate nucleic acid samples prepared according to thisinvention.

FIG. 3 is a plot of PCR assay results sequence and the second targetsequence with standard amounts of nucleic acid samples.

FIG. 4 is a perspective view of a container including a LysoDot.

FIG. 5 is a cross-sectional view of a chip containing a first chamberfor performing the first step of preparation according to this inventionand a second chamber for performing the second step according to thisinvention.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

Disruption Reagent according to this invention comprises a chaotropicagent, for example, a guanidium salt such as guanidine isothiocyanate orguanidine hydrochloride, or potassium iodide. Sodium hydroxide orpotassium hydroxide may be substituted as an equivalent only forpreparations of DNA alone, as that reagent destroys RNA. Our preferredchaotropic agent, guanidine isothiocyanate, tends to precipitate at lowtemperatures at concentrations of about 2 M and higher. Rather thanheating the reagent and the dispensing apparatus, such as a pipet, anon-aqueous, water-miscible solvent that evaporates during processing,such as dimethylsulfoxide (DMSO) can be included in the DisruptiveReagent in minor amount, for example 1% (vol/vol) to keep the reagentliquid during dispensing. The Disruptive Reagent additionally contains acell-lysing reagent, if the sample source may include intact cells. Weutilize 0.25% sarcosyl detergent, although others may be used as well,as will be understood by persons skilled in the art. Additionalingredients may optionally be included in the Disruption Reagent. Asindicated, our presently preferred Disruption Reagent includes 100 mMreducing agent, specifically βmercapto-ethanol, and 0.01 M neutralbuffer, specifically sodium citrate, pH=7.0.

As indicated, Disruption Reagent may be supplied for use either inliquid form or dry, as a LysoDot or LysoFilm as part of a container, incertain preferred embodiments. An example of an embodiment in which itmay be preferred to dispense Disruption Reagent in liquid form isstarting samples comprising cells in culture, such as cells grown inwells of microtiter plates. In that instance one may add liquidDisruption Reagent to the cells to form the reaction mixture. If thecells are relatively plentiful, say, numbering in the hundreds orthousands, one may reduce the mixture following disruption to a portionrepresenting at least one, preferably fewer than 100, cells. Either someof the mixture can be removed, leaving the desired portion in thecontainer for further processing, or the desired portion can be removedto a second container for further processing. The unused major portioncan be stored or subjected to other processing. Reduction to a portionby either technique can be carried out after the second step of nucleicacid preparation as well.

Methods according to this invention are particularly adaptable tosamples containing a small number of cells, for example a fraction of acell up to 200 cells or 1-100 cells, or equivalent amounts ofnon-cellular sample materials. Methods according to this invention areparticularly adaptable to processing that minimizes physical transfersof sample-containing mixtures, including performing nucleic acidpreparation and nucleic acid amplification and detection in a singlecontainer. In order to design methods according to this invention it isconvenient to calculate backwards. If a real-time assay is to beperformed, for example, in a 100 μl-assay container (currently availablethermal cycling instruments typically utilize 200 μl tubes that actuallyhave a 100 μl-assay capacity), one takes into account the dilutions tobe performed, either a single combined dilution or at least two andoften three serial dilutions. In the serial-dilution method describedabove, the final dilution occurs with the addition of amplification anddetection reagents, at least about a 9:1 dilution. The first dilutionoccurs in the second step of methods according to this invention andincludes a dilution of at least 50:1. There may be at least onesignificant intermediate dilution such as reverse transcription with adilution of up to 6:1, preferably not more than 2:1. Calculatingbackwards, one arrives at an appropriately small volume of DisruptionReagent for the first step of the method. The amount of DisruptionReagent is the amount of Disruption Reagent in liquid form before anydrying to prepare, for example, a LysoDot. Applying the foregoing designprinciple to our work with 200 μl PCR tubes and 100 μl assays, weselected a volume of Disruption Reagent of 20-50 nl as an appropriatestarting volume. Smaller volumes would, of course, be suitable as well.With guanidine isothiocyanate at a concentration of 2M, 20 nl ofDisruption Reagent is sufficient for up to about 100 cells.

Freeing nucleic acids is performed at a chaotropic agent concentrationof at least 2M. If the Disruption Reagent is added in liquid form, weprefer that the concentration of chaotropic agent be 2-8 M in theDisruption Reagent. If the Disruption Reagent is dried and rehydratedduring use, it is reconstituted to the same concentration range. Ifevaporation is utilized as part of the first step of the reaction, thevolume of Disruption Reagent added as a liquid or rehydrated from driedsolid can be increased, and the concentration of chaotropic reagentdecreased, prior to evaporation. The minimum 2 M concentration may beachieved through evaporation of water and volatile components, such asDMSO and βmercapto-ethanol. More preferred embodiments start withDisruption Reagent containing at least 2 M chaotropic agent to minimizetime of heating for evaporation.

In preferred methods, the first step includes incubation with heat toreduce the mixture of sample and Disruption Reagent to a wet or semi-drysolid or even a dry solid, taking care not to lose nucleic acids or atleast keep the loss below 25%. Such heating is preferred for threereasons. First, it concentrates the chaotropic agent to a very highconcentration, ensuring release of nucleic acids. Second, it reduces theincubation time needed and provides visual assurance that release hasbeen achieved. Third, it removes volatile agents that mightdeleteriously affect subsequent processing. For our laboratorypreparations, we typically open the tube at the end of the heating stepand place the lid holding the lysed sample under a microscope forinspection. That procedure would not be preferred for applications inwhich avoiding contamination is particularly important, such as inprocessing human samples.

The second step in methods according to this invention is a majordilution step. In certain preferred embodiments this occurs in the samecontainer. In embodiments in which the container includes flow channels,such as a chip, mixture from a first chamber, such as a well, in whichthe first step is performed, may flow into a separate chamber fordilution. In all cases, the second step is performed without firstseparating nucleic acids from degraded proteins. Except in certainembodiments in which one wishes to eliminate either RNA or DNA, thesecond step is performed without separating DNA molecules from RNAmolecules. The degree of dilution is at least 50:1 of the amount ofDisruption Reagent containing 2 M chaotropic reagent. Thus, if the firststep was performed with a volume of liquid Disruptive Reagent with 2 Mchaotropic agent, dilution is at least 50:1 on a volume-to-volume basis.If the first step was performed starting with a larger volume of LiquidDisruption reagent with 1 M chaotropic agent that was concentratedduring the first step, dilution is at least 25:1 on a volume-to-volumebasis, and so forth. Following dilution, the sample is prepared. Themixture is ready for further processing of nucleic acids, RNA or DNA orboth, without removal of degraded proteins and without removal ofDisruptive Reagent. Illustrations of use are set forth in the Examples.

Disruption Reagent may be supplied in dried form as part of a container,as discussed above. An embodiment is depicted in FIG. 4. For our ownlaboratory preparations, including Example 1 below, we have simply driedthe LysoDots in air, which is much slower than using a heating block asdepicted in FIG. 4, and the lysed samples on a rack placed in a waterbath maintained at about 75° C. FIG. 4 shows a container 41, a PCR tube,more specifically a 200 μl PCR tube. Container 41 comprises body 42 andsealing cap 43. Dried Disruption Reagent, in the embodiment LysoDot 44,is adhered to cap 43. Preparation of the container part 43 with adheredLysoDot 44 was as follows: a small amount, in our case, 20 nl ofDisruption Reagent was added to cap 43 resting in depression 45 of heatblock 46. The Disruption Reagent was heated until it dried to LysoDot44, adhering to cap 43. LysoDot 44 could equally be placed in body 42and dried there. We chose to put it into cap 43 only because of the easeof pipetting Disruption Reagent by hand onto the center of cap 43. Wehave carried out the first step of methods according to this inventionin cap 43, by adding thereto a sample, for example, embryonic cells inisotonic solution, namely phosphate-buffered saline (PBS), therebyrehydrating the dried Disruption Reagent. Tube 41 may then be closed forincubating and evaporating the mixture to a semi-dry or dry solid. Atthis point the mixture can be used immediately or it can be frozen orrefrigerated. Heating/evaporation is carried out under conditionsavoiding cross-contamination of samples. Closing tube 41 is one way toavoid that occurrence. Other ways will be apparent to persons skilled inthe art.

Next, the fifty-fold dilution solution is added to cap 43, having openedtube 41 if necessary. At this point it is desirable to utilize tube 41right-side up, so we reseal the tube and cause the mixture to move totube body 42. Centrifugation may be used to facilitate the transfer.Subsequent processing is carried out in the tube in its uprightposition.

Another LysoDot- or LysoFilm-containing apparatus is shown in FIG. 5.FIG. 5 depicts an apparatus 50 through which reagents are moved undercontrol during processing. Apparatus 50 may be, for example a chiphaving wells and chambers. Chip 50 includes a well 59 containing driedDisruption Reagent, in this case LysoFilm 518. A sample may be added towell 59, which can then be closed with cap 52. The sample, nowcontaining liquefied Disruption Reagent, could be moved to chamber 510through channel 519, controlled by means not shown, by pressure appliedthrough port 57 or 58, for incubation, drying and dilution in the secondstep. Chamber 510 is connectable to a source of dilution reagent bymeans of channels 54, 56, 514. Alternatively, incubation and heating(drying) could be carried out in well 59, after which the mixture couldbe liquefied and delivered to chamber 510 by means of all or some of thedilution reagent. The prepared mixture could then be delivered, bycontrollable means not shown, or by pressure applied through port 57 or58 to one or more chambers 511, 512, or 513, etc for further processing.Chambers 511, 512, 513 are connectable to reagent supply sources byadditional channels, for instance 515, 516. A fraction of the materialin chamber 511 can be removed by channel 55. Positive gas pressure inthe system can be released through channel 517 that contains a filter523 to prevent the escape of non-gaseous molecules. In one design, theblock, 51, in which these channels and wells are built is comprised of amaterial that can rapidly heated and cooled, and in the most preferredcase can be heated and cooled be differently for different wells andchambers. In another design, the block 51 is comprised of insulatingmaterial that is not easily heated or cooled, and temperature changesare achieved through at least one surface of each chamber, by means notshown. In yet another design, the entire block 51 is cooled continuouslyand the temperature is raised by means not shown, for example byresistance heating, through the surface of each chamber or by passinginfrared light into each chamber. In preferred designs at least onesurface of each chamber is transparent to permit transmission of lightinto the chamber. In preferred designs all surfaces within the chamberare made from or is coated with inert materials which do not bindnucleic acids or other chemicals or proteins. In preferred designs thechambers and channels are designed to minimize turbulence or drag offlowing liquids. Numerous variations of LysoDot preparations areincluded in this invention. In addition to removable caps or lids,LysoDots can be adhered to any surface suitable for use in carrying outthe first method step, for example, attached lids or bodies of PCRtubes, microscopy slides or cover slips, wells of multiwell trays. TheDisruption Reagent may also be supplied as dried pellet or as acomposition dried onto a solid support, such as a fiber or matrix, orbead, that is inert and does not bind nucleic acids. For purposes ofthis application such a solid support is considered a container orcontainer part for appropriate use.

Numerous variations are possible following the first step in methodsaccording to this invention. The incubation of the first step mayinclude heating to dry the mixture, after which it can be stored frozenat, for example, −20° C., for long periods or even stored at roomtemperature for significant periods. Further, use of the mixtureresulting from the first incubation step of preparation may proceed by aseries of separate steps, as outlined above, or two or more steps may becombined. For example, reverse transcription and amplification reagentsmay be included in a single reagent that dilutes by at least about afactor of nine. The second step, at least fifty-fold dilution, can becombined with subsequent steps in appropriate cases. It will beappreciated that thermostable enzymes are required for processing athigh temperatures needed, for example, for strand melting.

If it is desired to utilize RNA only, DNA can be degraded by a DNAendonuclease, for example, DNase I. Degradation may follow the secondstep of purification, or DNA endonuclease may be included in thedilution reagent used in the second step, together with salts requiredfor the activity of the DNA endonuclease that is used. Followingelimination of DNA, a chelating agent is added at a concentrationsufficient to prevent magnesium-dependent hydrolysis of RNA, and themixture is heated, for example for 10 minutes at 65° C., to inactivatethe endonuclease prior to reverse transcribing RNA to cDNA.

Particularly for applications such as microarray analysis, we haveutilized a DNase digestion step preceding reverse transcription in oursingle-tube format for Xist/Sry analysis reported below in Example 1. Weanalyzed the efficiency with which genomic DNA in morula-stage mouseembryos was degraded. In the fifteen male samples analyzed, wedetermined that 96 percent of the DNA had been degraded. In the tenfemale samples analyzed, RNA recovery was lower than in non-treatedsamples. Some loss of RNA by a DNase step was expected. Our RNArecovery, however, was considerably higher than we obtained withtraditional RNA extraction using either a Micro RNA Isolation Kit(Stratagene, La Jolla, Calif., U.S.A) or an RNeasy Mini Kit (Qiagen,Inc., Valencia, Calif., U.S.A.) coupled to DNase digestion. Other enzymetreatments may be included in or following the second step as may beappropriate. For example, one may digest plant cell walls or othercomponents using a glycosidase enzyme, or one may digest bacterial cellwalls using lysozyme.

Another processing variation is to avoid reverse transcription for allor a portion of a prepared mixture. For example, a mixture resultingfrom the second step may be divided into two portions. One portion maybe further processed by RT-PCR to quantify total cDNA plus genomic DNA.The other portion may be further processed without reverse transcriptionsuch that only genomic DNA is amplified. Reverse transcription can beavoided in any suitable manner, including by omitting reversetranscriptase, by omitting primers for reverse transcription, or bydestroying RNAs with an RNase.

Division of mixture following the second step or later in processing maybe done for other reasons. For example, rather than performing amultiplex assay with multiple primer pairs and multiple hybridizationprobes, a mixture may be divided for parallel non-multiplex assays.

Other processing variations may also be used. For example, RNAs may beprepared according to this invention, labeled directly or asreverse-transcribed cDNA, and subjected to hybridization and analysis ona microarray of immobilized oligonucleotides.

EXAMPLES Example 1 Assays.

Sample preparation was performed according to this invention, followedby real-time multiplex PCR assays, all in a single tube. Processingsubsequent to preparation, that is, following the second step, utilizedthe assay materials and methods reported in Hartshorn, C. et al (2002),Developmentally-Regulated changes of Xist RNA Levels in SinglePreimplantation Mouse Embryos, as Revealed by Quantitative Real-TimePCR, Mol. Reprod. Dev. 61: 425-436; and Hartshorn, C. et al (2003),Differential Patterns of Xist RNA Accumulation in Single BlastomeresIsolated from 8-Cell Stage Mouse Embryos Following Laser Zona Drilling,Mol. Reprod. Dev. 64: 41-51, both of which are incorporated herein intheir entirety. Six mouse embryos were grown to the blastocyst stage, asdetailed in the first paper cited above. All experimental procedureswere carried out rigorously following precautions aimed at avoiding ordestroying environmental RNase contamination as described in both paperscited above. LysoDots were prepared several days before embryocollection by delivering 20-nl aliquots of Disruption Reagent to thelids of reaction tubes. Precise measurement of droplet size was obtainedfollowing the method previously described by Wangh (Wangh, L. J. (1989),Injection of Xenopus Eggs Before Activation, Achieved by Control ofExtracellular Factors, Improves Plasmid DNA Replication afterActivation, J. Cell Sci. 93:1-8). The Disruption Reagent compositionwas:

-   -   0.25% Sarcosyl    -   2 M Guanidine Isothiocyanate    -   100 mM β Mercapto-ethanol    -   1% (vol/vol) Dimethylsulfoxide    -   0.01 M Sodium Citrate, =pH 7.0

LysoDots of 20 nl were allowed to dry by placing open tubes under ahood, in sterile conditions. Most droplets dried overnight, but somerequired 48 hours for crystallization to occur. No difference wasobserved among these samples in terms of experimental results. Tubeswere closed and stored upside down at room temperature until use.

Immediately before harvesting, individual embryos were placed in 3 ml ofDulbecco's PBS. Each embryo was then aspirated into a glass capillaryhaving an internal diameter of 0.2 mm and tapered at the end so that theinner volume of the tapered tip would contain about 20 nl. Tapering wasobtained by pulling the glass capillaries in a Micro-Pipette Puller(Industrial Science Associates, Inc., Ridgewood, N.Y.). A blunt conicalend was then produced by gently removing the thinnest portion of the tipwith an abrasive stone. For an optimal delivery of the embryo to theLysoDot, PBS was aspirated into the pipette slightly beyond the taperedend. The embryo was allowed to move toward the pipette's tip and wasthen expelled directly onto the dry LysoDot in a volume of PBS that wasas close as possible to 20 nl (some PBS was left in the pipette). Thiswhole procedure was carried out under a microscope, so that the embryo'sposition in the pipette could be monitored. Microscopic observation alsomade it possible to check for an immediate and complete dissolution ofthe LysoDot crystals upon addition of the embryo-containing PBS.

Tubes were closed upside down and transferred in this position to a rackplaced in a closed bath heated to 75-77° C. Incubation was carried outfor 5 minutes. After this step, the sample-containing mixtures weremostly dry or partially dry, due to evaporation. They were stored upsidedown at −20° C. until the next step.

The second step of preparation was next carried out. In this case,because the example includes reverse transcription, the dilution reagentcontained oligonucleotides in addition to water. The tubes were openedagain under a microscope and each dry droplet was re-solubilized byaddition of 6 μl of a mixture of Random Hexamers (4.2 ng/μl) inDEPC-treated water (all reagents were from a ThermoScript™ RT-PCR Systemkit, Invitrogen, Life Technologies, Carlsbad, Calif., USA). Tubes wereclosed and briefly placed, still in the upside down position, on avortex. Samples were then centrifuged, mixed again on the vortex(right-side up) and spun down one more time.

The closed tubes containing the prepared nucleic acid samples wereheated to 65° C. for 5 min to melt double-stranded RNA molecules andthen cooled to hybridize the oligonucleotide primers to sequences withinthe RNA molecules. Then the tubes were opened for addition of 4 μl ofreverse transcriptase solution, which diluted the mixture by a factor ofabout 1.7 volume increase. The solution contained 0.5 μl of ThermoScriptreverse transcriptase (15 U/μl), 1 μl of 10 mM dNTP Mix, 0.5 μl ofRNaseOUT™ (40 U/μl) and 2 μl of 5× cDNA Synthesis Buffer (250 mM Trisacetate, pH 8.4, 375 mM potassium acetate, 40 mM magnesium acetate). Thetubes were closed and incubated for 10 min at 25° C. followed by 50 minat 55° C. to prepare cDNA copies of RNA. The reaction was terminated byheating the closed tubes for 5 min at 85° C., which denatured thereverse transcriptase. Next, the tubes were cooled on ice, centrifugedbriefly and opened for addition of 0.5 μl of E. coli RNase H (2U/μl).Incubation was carried out for 20 min at 37° C. in order to degrade theRNA molecules present in the sample. Samples were cooled and centrifugedagain, and stored at 20° C. until the next step.

In one experiment, the full volume of each sample was transferred to aPCR tube where it was mixed with 89.5 μl of complete PCR amplificationand detection reagents. The reagents comprised 54.3 μl of PCR-gradewater, 10 μl of 1 M Tris chloride, pH 8.3, 16 μl of 25 mM magnesiumchloride, 4 μl of 10 mM dNTP (2′-deoxynucleoside 5′-triphosphates) Mix,3 μl of a 10 μM mix containing the upper and lower primers foramplification of a sequence of DNA and RNA from the gene Xist and theupper and lower primers for amplification of a sequence of the gene Sry,molecular beacon probes for the amplicon of the Xist gene and for theamplicon of the Sry gene (0.3 μl each of 100 μM solutions), and 1.6 μlof a mix containing 4 U of Taq polymerase (Promega, Madison, Wis.)complexed to Platinum Taq antibody (Invitrogen, Carlsbad, Calif.) bymeans of a 5 min incubation at room temperature prior to addition to thePCR solution. Amplification and detection were carried out in an ABIPrism 7700 Sequence Detector. The cycling profile was: 95° C. for 5 min;10 cycles consisting of the following three steps: 95° C. (20 sec), 57°C. (60 sec), 72° C. (30 sec); 45 cycles with the following three steps:95° C. (20 sec), 53° C. (60 sec), 72° C. (30 sec).

Assay results are shown in FIGS. 1 and 2. Referring to FIG. 1, it can beseen that three embryos (Circle 11) had an Sry signal and, therefore,were male, while the other three embryos (Circle 12) had no Sry signaland, therefore, were female.

It is known that female blastocysts express high levels of Xist RNA,responsible for inactivation of one X chromosome, while male embryoshave virtually no Xist. The results obtained with preparation accordingto this invention fully confirmed this expectation. Referring to FIG. 2,it can be seen that all three female embryos (Circle 21) generatedsignals detectable above background approximately six thermal cyclessooner than the male embryos (Circle 22).

As indicated above, no processing of samples was performed betweenreverse transcription and PCR. The results demonstrate the effectivenessof this invention to directly prepare samples for amplification andfurther demonstrate that all processing could be performed in a singletube. In other experiments all steps were performed successfully in asingle PCR tube to the cap of which we had initially added a LysoDot.For example, we have used the single-tube assay on the followingsamples: two-, three- four-and eight-cell mouse embryos and mouseembryos at the blastocyst stage, single blastomeres isolated from fourand eight-cell mouse embryos, and mouse embryos treated with DNase (seeExample 3 below).

Example 2 Quantitation.

Control reactions containing decreasing amounts of male mouse genomicDNA (10,000 genomes down to 10 genomes) were used to construct linearplots of Genome Number vs. C_(T), the thermal cycle at which signalbecomes detectable above background. Results are shown in FIG. 3. Theplots for both the Xist (Curve 31) and Sry (Curve 32) signals fall onstraight lines that virtually overlap. Comparison of the C_(T) values ofthe curves of Circle 21 in FIG. 2, which have a C_(T) of about 19, withstandard curve 31 of FIG. 3 and the curves of Circle 22, which have aC_(T) of about 25, with standard curve 31 of FIG. 3, allow us tocalculate that each embryo contains about 100 genomes and each femaleembryo contains several thousand copies of Xist RNA (cDNA). Theseresults are very similar to our previous findings, reported in theabove-identified papers, based on a traditional approach to nucleicacids extraction from mouse embryos.

None of the male embryos assayed with the sample preparation method ofthis invention as part of a single-tube assay had levels of Xist DNAgreater than the levels of Sry DNA. This is seen by comparing the C_(T)values of the curves in Circle 11 (FIG. 1) with the C_(T) values of thecurves in Circle 22 (FIG. 2). This result is consistent with the absenceof Xist gene expression in male embryos and the presence of one Xchromosome (one Xist gene) and one Y chromosome (one Sry gene) in eachmale cell. Two of the male embryos have about 100 genomes, as expected,while the third male embryo was probably composed of fewer cells (afinding not unusual for cultured embryos). In the female embryos thepresence of Xist RNA in addition to Xist genomic DNA leads to thedetection of thousands of Xist template copies in each sample.

Example 3 DNase Treatment.

The efficiency of DNase treatment in samples prepared with the method ofthis invention was tested as follows.

Thirty-five mouse embryos at the morula stage (about 16-32 cells) werecollected individually on LysoDots and disrupted as detailed for Example1, the difference being that embryos were purchased frozen at the 8-cellrather than the 2-cell stage. Each assay was carried out from beginningto end in the same 200 μl PCR-grade tube (microAmp Optical Tubes,Applied Biosystems, Foster City, Calif.), although the lids (OpticalCaps, Applied Biosystems) were replaced every time the tubes were openedin order to avoid contamination. Ten samples were processed completelyas in Example 1, thus containing both genomic DNA and cDNA at the end ofthe assay, and were designated as controls. In the remaining 25 samplesthe second step of preparation was modified to allow genomic DNAdegradation, as explained below, so that in this protocol reversetranscription became the third step of preparation and PCR the fourth.Particular care was taken in assigning embryos with equivalentdevelopmental and morphological features to the Control and theDNase-Treated groups.

Each disrupted and heated sample (dry or partially dried) wasresuspended on the lid originally containing the LysoDot with 4 μl ofDNase Mix having the following composition:

DNase Mix (reagents ration for 10 μl):

1 μl 10 X Reaction Buffer (Invitrogen, Life Technologies, Carlsbad, CA)containing 200 mM Tris-HCl, pH 8.4, 20 mM MgCl₂, and 500 mM KCl 0.5 μlDNase I (2 U/μl) (Ambion, Inc., Austin, TX) 8.5 μl Nuclease-free H₂O(Ambion)

A 200-fold dilution of the reagents originally comprising the LysoDotwas thus achieved in this step, bringing the concentration of guanidinein the DNase digestion step to 10 mM (2 M diluted 200:1), at whichconcentration the enzymatic reaction was not inhibited.

Tubes were closed, vortexed in the upside-down position, inverted andcentrifuged as described in Example 1. Digestion of the genomic DNA inthe samples was carried out for 20 minutes at room temperature, afterwhich the reaction was terminated by adding 1 μl/assay of a 10 mM EDTAsolution (molecular biology grade, Invitrogen) and heating at 65° C. for10 minutes. As known to those skilled in the art, the addition of achelating agent is needed in order to avoid magnesium-dependent RNAstrand scission during the heating step which denatures the DNase,therefore blocking its further activity when cDNA is produced during thereverse transcription of RNA.

After DNase treatment, each sample received 2 μl or Random HexamerPrimer Mix prepared as follows:

0.5 μl Random Hexamers (50 ng/μl) 0.5 μl DEPC-treated H₂O

(Both reagents from Invitrogen, as described in Example 1.)

The total volume of each assay at this point was 6 μl. After incubationof the primers with RNA, as detailed in Example 1, reverse transcriptionreagents were added in a volume of 4 μl and all the remaining steps ofreverse transcription and PCR were also carried out as described inExample 1.

The results indicated that, in the average, 42.6 copies per embryo ofgenomic Xist plus Sry DNA were recovered from male control samples,corresponding to an average number of 21.3 cells per embryo andconfirming the expected cell number for embryos at the morula stagebased on the fact that one male cell contains one copy of each of thesetwo genes. After DNase treatment, only 1.6 copies per embryo of genomicXist plus Sry DNA were left in the male samples, or 4% of the controlvalue, indicating that DNase activity in the presence of 10 mM guanidineand under the experimental conditions utilized was optimal and degraded96% of the DNA present. (In a parallel experiment, DNase incubationcarried out for 15 rather than 20 minutes resulted in the degradation of90% of genomic DNA in male samples.) This level of effectiveness wasjudged to be very high, because it is known that DNase I cleaves DNA atlow concentration rather inefficiently, having poor affinity for itssubstrate.

Not unexpectedly, RNA recovery was partially affected by the presence ofthe DNase step. As widely debated in the literature, there is no idealmethod allowing complete DNA digestion without altering RNA recovery.

In this assay, the average Xist RNA recovery from DNase-treated femalesamples was lower than the Xist RNA recovery from female controls. Theamplification of DNA templates during PCR was unaffected by the presenceof the DNase Mix reagents.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.

1-23. (canceled)
 24. A device for use in processing a biological sample containing protein-bound RNA and DNA molecules to free said molecules from proteins comprising a dried disruption reagent comprising a chaotropic agent adhered to a surface of a container or container part.
 25. The device according to claim 24, wherein said container or container part is selected from the group consisting of a tube, a tube cap, a microtiter plate, and a cover for at least one well of a microtiter plate.
 26. The device according to claim 25, wherein said container or container part comprises multiple chambers connected by flow channels.
 27. The device according to claim 24, wherein said dried disruption reagent comprises an amount of chaotropic agent that will produce a concentration of 2M-8M chaotropic agent when dissolved in 20-50 nl of water.
 28. The device according to claim 24, wherein said dried disruption reagent comprises a detergent.
 29. The device according to claim 24, wherein said dried disruption reagent comprises at least one component selected from the group consisting of a reducing agent, a chelating agent, a water-miscible solvent, and a buffer.
 30. A kit comprising at least one reagent useful for enzymatic processing of nucleic acids and a device according to claim
 24. 31. The kit according to claim 30, comprising primers and enzyme for reverse transcription.
 32. The kit according to claim 31, comprising a DNase enzyme.
 33. The kit according to claim 30, comprising nucleic acid amplification reagents comprising at least DNA polymerase and amplification buffer.
 34. The kit according to claim 33, comprising at least one pair of polymerase chain reaction primers and at least one sequencing primer.
 35. The kit according to claim 33, comprising at least detection reagent.
 36. The kit according to claim 35, comprising at least one dual labeled fluorescent hybridization probe.
 37. (canceled)
 38. (canceled) 